Soluble CD4 effectively prevents excessive TLR activation of resident macrophages in the onset of sepsis

T lymphopenia, occurring in the early phase of sepsis in response to systemic inflammation, is commonly associated with morbidity and mortality of septic infections. We have previously shown that a sufficient number of T cells is required to constrain Toll-like receptors (TLRs) mediated hyperinflammation. However, the underlying mechanisms remains unsolved. Herein, we unveil that CD4+ T cells engage with MHC II of macrophages to downregulate TLR pro-inflammatory signaling. We show further that the direct contact between CD4 molecule of CD4+ T cells or the ectodomain of CD4 (soluble CD4, sCD4), and MHC II of resident macrophages is necessary and sufficient to prevent TLR4 overactivation in LPS and cecal ligation puncture (CLP) sepsis. sCD4 serum concentrations increase after the onset of LPS sepsis, suggesting its compensatory inhibitive effects on hyperinflammation. sCD4 engagement enables the cytoplasmic domain of MHC II to recruit and activate STING and SHP2, which inhibits IRAK1/Erk and TRAF6/NF-κB activation required for TLR4 inflammation. Furthermore, sCD4 subverts pro-inflammatory plasma membrane anchorage of TLR4 by disruption of MHC II-TLR4 raft domains that promotes MHC II endocytosis. Finally, sCD4/MHCII reversal signaling specifically interferes with TLR4 but not TNFR hyperinflammation, and independent of the inhibitive signaling of CD40 ligand of CD4+ cells on macrophages. Therefore, a sufficient amount of soluble CD4 protein can prevent excessive inflammatory activation of macrophages via alternation of MHC II-TLR signaling complex, that might benefit for a new paradigm of preventive treatment of sepsis.


INTRODUCTION
Innate immune cells sense microbial pathogen-associated molecular patterns (PAMPs) via pattern recognition receptors (PRRs), which include Toll-like receptors (TLRs), NOD-like receptors (NLRs), C-type lectin receptors (CLRs) and nucleic acid sensors. [1][2][3] The recognition upregulates both major histocompatibility complexes (MHC I and II) and co-stimulatory molecules, and secretes inflammatory cytokines, to prime subsequent adaptive immune response for protection. 4 Hyperinflammation due to excessive activation of innate immune cells, however, leads to pathological alterations and correlates with the morbidity and mortality of infections. 5 Innate inflammation therefore has to be tightly controlled by a series of negative regulators, at multiple levels, to maintain immunological homeostasis. 6,7 We established previously that a sufficient amount of naïve T cells is required to dampen the acute innate inflammatory response to betacoronavirus and Gram-negative bacteria infections. 8,9 To do so, T cells need to make direct cell-cell contact with antigen presenting cells (APC) to suppress their TLRmediated inflammatory response, but independent of T cell receptor (TCR). 8 Intriguingly, both effector and memory CD4 + T cells can block caspase-1/IL-1β activation by cell-cell contact with APC that diminishes NLRP3 and NLRC4 inflammasome activation. 10 Sepsis is defined as an initial hyper-inflammatory response to systemic infection associated with a subsequent immune suppression and dysfunction, that can lead to multiple organ failure, secondary infections and mortality. 11 The onset of sepsis is characterized by leukocytosis (marked increase in neutrophils and monocytes) in the first 2-4 days, followed by a state of lymphopenia as a result of apoptosis (drastic reduction of B cells, CD4 + and CD8 + T cells). Failure to restore cell numbers during either the stage of leukocytosis or lymphopenia results in increased mortality. 12,13 Sepsis patients develope long-term immune impairment due to the reduction in the number and function of different immune cell populations. 14 Patients who survive the early phase of sepsis develop immune suppression, which is characterized by T cell exhaustion, unresolved infection and susceptibility to opportunistic infection, and dysfunctional T cell repertoire. 13 Compared to the understanding of functional impairment, however, the pro-apoptotic factors that cause T lymphopenia in sepsis and the impact of T lymphopenia to sepsis remain elusive. 15 T lymphopenia, responding to systemic inflammatory response in the early phase of sepsis, 16 is commonly associated with morbidity and mortality of septic infections. 17 Such an acquired T-lymphopenia also occurs in certain autoimmune diseases and inflammageing process. 18 Critically, insufficient number of naïve CD4 + T cells is responsible for the unleashed TLR inflammation with high morbidity and mortality rates. 3 CD4 uses its extracellular domain to interact with β 2domain of MHC II, 19,20 and MHC II is required for CD4 + T cells to suppress TLR hyperinflammation in APCs. 8,9 Intriguing enough, MHC II is required to constrain B cell-response to LPS, 21,22 and decreased expression of HLA-DR in monocytes and macrophages is observed in sepsis patients. 23 Nevertheless, how CD4 + T cells signal through MHC II to suppress TLR inflammation in APCs remains unknown.
In this study, we demonstrate that CD4 molecule, either membrane bound or soluble ectodomain (sCD4), is necessary and sufficient to hamper TLR inflammation of APC and protect mice from the lethal septic inflammation. Furthermore, CD4 engagement recruits SHP2 and STING to the intracellular tail of MHC II in lipid rafts, which disrupts MHC II/TLR4 signaling complex, and inhibits TLR4 activation of IRAK1/TRAF6/NF-κB inflammatory response.

RESULTS
CD4 T-lymphopenia accounted for the morbidity and mortality of sepsis We used LPS endotoxemia in mice as a surrogate of sepsis in patients 24 to study the role of CD4 + T lymphopenia. C57BL/6 mice succumbed within 72 h post LPS i.p.injection (hpi, 10 mg/kg. Fig.  1a), accompanied with severe numerical reduction of both CD4 + (Fig. 1b) and CD8 + T cells ( Supplementary Fig. 1a) in the thymus and periphery at 12 hpi, with the thymus drastically shrunk (Supplementary Figs. 1b,. CD4 + T cells were more prone to sepsis-induced apoptosis, shown by reduced ratios of CD4/CD8 T cell counts in the thymus and spleen, as compared to the sham mice ( Supplementary Fig. 1c). T lymphopenia was also observed in septic infection of mice by oral gavage of Salmonella typhimurium SL1344 ( Supplementary Fig. 1d). To substantiate that LPS sepsis induced T lymphopenia can recapitulate that in the classical cecal ligation and puncture (CLP) sepsis (Fig. 1c), we compared side-byside two models in the first 24 h after treatment. Both models showed qualitatively and quantativevly similar reduction of thymic sizes ( Supplementary Fig. 2a) and increased pathology scores (Supplementary Table 4), as reflected by decreased thymocyte numbers, abnormal proportion of medulla and cortex and increased cell death ( Supplementary Fig. 2b, c). Both models showed similar tendencies of T cell number reduction, decreased CD4/CD8 ratios except that in thymi of CLP model ( Supplementary  Fig. 2d,e). These results agreed with the previous CLP results. 15 Therefore, T lymphopenia induced by a septic dose of LPS in mice manifested the dysregulated T cell response in CLP sepsis.
Numerical and functional dysregulation of CD4 + and CD8 + T cells plays important roles in sepsis onset, progression and recovery. 12,13 Insufficient number of CD4 + T cells per se led to the hyperinflammation and mortality in sepsis, because mice lacking CD4 + T cells, either by antibody-ablated (Fig. 1g) or in nude mice (Fig. 1h), were more susceptible to LPS sepsis (poorer survival and higher TNF/IL-6), compared to those by isotype antibody treatment ( Supplementary Fig. 1i for antibody depletion efficiency) or CD4 + T cells adoptive transfer ( Supplementary Fig. 1j for adoptive transfer efficiency), respectively. C57BL/6 mice of CD4 + T cells pre-depleted also showed augmented TNF/IL-6 response to SL1344 oral infection ( Supplementary Fig. 1k) and much higher bacterial load ( Supplementary Fig. 1l). Likewise, antibody depletion of CD4 + T cells in K18-hACE2 mice caused higher TNF/IL-6 expression (Fig. 1i) and more severe lung pathology (Fig. 1j, k) 7 d post SARS-CoV-2 infection. Therefore, aside from the functional exhaustion of T cells in the later stage of sepsis, the drastic reduction of CD4 + T cell numbers would attribute to the first hit of systemic hyperinflammation of sepsis.
To test critically whether CD4 molecule per se directly mediates CD4 + T-cell suppression of LPS/TLR4 inflammation, isolated T cells were co-cultured with bone marrow derived macrophages (BMDM) 2 h before LPS stimulation. T cells lacking CD4, either foreign antigen naïve (thymus DNT) or experienced (splenic DNT), failed to inhibit LPS activated TNF/IL-6 expression, except for naive or thymic single-positive CD4 + T cells (Fig. 1l). Therefore, CD4 moiety of naïve CD4 + T cells, but not TCR or TCR restriction as indicated previously, 8 played an essential role to suppress TLR4 inflammation. This notion was substantiated by the co-culture experiments that anti-CD4 antibodies (RM4-5 or GK1.5) completely prevented CD4 + T cells from inhibiting TLR4 inflammation in macrophages (Fig. 1m). Of note, antibodies did not induce CD4 + T cell apoptosis ( Supplementary Fig. 1m). To exclude the involvement of other CD4 + T-cell components, e.g., co-stimulatory receptors, we co-cultured THP-1 cells with TZM-b1 cells, a HeLa derivative that stably expresses human CD4. 27 Only TZM-b1, but not HeLa cells, prevented THP-1 cells from TNF and IL-6 response to LPS (Fig. 1n). Collectively, CD4 molecule was necessary and sufficient to restrain TLR4 inflammatory response.
Acute LPS inflammation causes bone marrow (BM) monocytes to rapidly egress into circulation and infiltrate tissues, where they differentiate to inflammatory DCs and macrophages. 28 Careful comparison of nude or nude mice adoptively transferred with CD4 + T cells ( Supplementary Fig. 3a for the partial BM reconstitution) showed that, CD4 + T cell deficiency led to more Ly6C hi monocytes retained in BM and less migrated to the blood or infiltrated to the spleen and liver 3 hpi (Fig. 2a, Supplementary  Fig. 3b for gating strategy). This can be partially explained by the inhibitive effect of CD4 + T cells on TLR4 inflammation-driven emergency myelopoiesis in BM (reduced hematopoietic stem cells, common myeloid and granulocyte macrophage progenitors), with megakarayocyte and erythrocyte progenitors intact (Fig. 2b, Supplementary Fig. 3c for gating strategies of hematopoietic lineages). Lack of CD4 + T cells also suppressed monocytes activation (Ly6C hi CD64 + ) in BM, the blood, and other indicated peripheral organs (Fig. 2c), likely due to insufficient IFNγ in the absence of CD4 + T cells ( Supplementary Fig. 3e), as suggested previously. 29 These results suggested that CD4 + T cells promoted emergency myelopoiesis and monocyte migration to inflamed tissues, necessary for inflammatory leukocytosis. In vitro co-culture experiments showed that CD4 + T cells accelerated TLR4-driven BM monocytes differentiation to macrophages, but DC significantly suppressed (Fig. 2d). Indeed, differentiation of spleen/liverinfiltrated monocytes to macrophages (CD11b int F4/80 -MHC II + , Supplementary Fig. 3d for gating strategies) were increased by CD4 + T cells in endotoxemic mice (Fig. 2e), but not the activation of these monocyte-derived macrophages (CD86 + , Fig. 2f). Surprisingly, the number of resident macrophages (CD11b low F4/ 80 hi ) in the spleen or liver remained unaffected by the presence or absence of CD4 + T cells (Fig. 2g), but their activation by LPS was effectively inhibited by CD4 + T cells (Fig. 2h). These results would suggest that resident macrophages played a more important role in mediating hyper-inflammatory response to CD4 + T lymphopenia in sepsis. qPCR measurement showed that TNF production was specifically reduced in splenic resident macrophages, but not infiltrated macrophages or monocytes, isolated 3 hpi (Fig. 2i).
Therefore, CD4 + T cells would play profound roles in promotion of inflammatory leukocytosis and prevention of unleashed TLR4 inflammation of resident macrophages.
CD4/MHCII engagement suppressed macrophage inflammation MHC II is required for T cells to hamper innate inflammation. 8 Specifically, MHC II transmitted the inhibitive signal of CD4 to TLR4 activation, because neither isolated CD4 + T cells (Fig. 4a) nor sCD4 ( Fig. 4b) were able to prevent LPS activation of MHC II-deficient (MHC II −/− ) macrophages in vitro, or in MHC II −/− mice (Fig. 4c, d). CD40L of CD4 + T cells can also downregulate macrophage TNF response to LPS through a paracrine activation of IL-10. 34 This process did not require MHC II, because trimeric CD40L protein (sCD40L) still inhibited TNF/IL-6 response to LPS in MHC II −/− macrophages in vitro (Fig. 4e). On the other hand, sCD4 inhibited LPS inflammation as efficient in CD40 -/-BMDM as in wt macrophages (Fig. 4f). Therefore, CD4 and CD40L of CD4 + T cells would function independently to prevent APC from TLR overactivation.
To critically test whether MHC II in macrophages per se is sufficient to mediate sCD4 antiphlogistic effect, macrophages were first depleted by clodronate liposome (Supplementary Fig.  5a). Macrophages depleted mice (ΔMΦ) partially resisted to lethal dose of LPS challenge (Fig. 4g), accompanied with reduced TNF/IL-6 ( Fig. 4h). Consequently, sCD4 inhibited TLR4 inflammation only when MHC II +/+ (Fig. 4i), but not MHC II ─/─ (Fig. 4j) macrophages were adoptively transferred to ΔMΦ mice. To further determine which subunit of MHC II was required to transmit the reverse signal of sCD4, different truncational mutants of MHC II subunits fused with eGFP were overexpressed in isolated peritoneal macrophages (Supplementary Fig. 5b for equal ectopic expression). Deletion of the cytoplasmic tails of MHCII A/E-α and A-β abrogated the inhibitive effect of sCD4 on TNF/IL-6 response ( Fig.  4k). Therefore, these results suggested that MHC II in macrophages was sufficient to transmit CD4 reverse signal to suppress TLR hyperinflammation.
SHP2 was required to mediate MHC II crosstalk to TLR pathway LPS is sensed by TLR4/MyD88, which recruits and phosphorylates IRAK1 to activate MAPK and NF-κB signaling. 35 The previous results show that the cytoplasmic tail of MHC II can contact MyD88 to inhibit TLR4 signaling. 36 We then set to test whether sCD4 engagement of MHC II would take advantage of the same pathway to crosstalk to TLR pathway. Phosphorylation of IRAK1 in BMDM (Fig. 5a), as well as Erk, Jnk, p38 and IκBα ( Fig. 5b and Supplementary Fig. 6a), in response to LPS was effectively inhibited by sCD4. Importantly, sCD4/MHC II ligation antagonized specifically the TLR signaling, because TNFR mediated MAPK or NF-κB activation was not affected (Fig. 5c). There are few reports that IRAK1 phosphorylation can trigger its proteosomal degradation, that may help reducing TLR4/NF-κB activation. 37,38 The total IRAK1 protein level slightly decreased even after a longer time of LPS stimulation (Fig. 5a, g). More experiments are needed to determine the role of IRAK1 degradation in MHCII reverse signaling.
Tyrosine phosphorylation plays an important role in the negative regulation of TLR signals. 39 The global tyrosine phosphorylation in LPS-treated BMDM was increased by sCD4 ( Supplementary Fig. 6b), where SHP2 phosphorylation (Fig. 5d), but not Btk, Syk or SHP1 ( Supplementary Fig. 6c), was specifically enhanced. SHP2 is a protein tyrosine phosphatase that regulates MyD88 signaling by contact with TRAF6. 40 LPS-induced binary interaction between SHP2 and TRAF6 was augmented by sCD4, as measured by reciprocal co-immunoprecipitation assays (Fig. 5e). Therefore, SHP2 would mediate CD4/MHC II crosstalking to TLR/ MyD88 pro-inflammatory signaling. Indeed, sCD4 failed to inhibit TNF/IL-6 response to LPS in SHP2-deficient BMDM (Fig. 5f), where IκBα (NF-κB) and IRAK1 (MAPK) activation was restored (Fig. 5g). In mice with SHP2 specifically ablated in macrophages, sCD4 no long improved the survival rates of LPS sepsis (Fig. 5h), nor inhibited TNF/IL-6 response (Fig. 5i). Besides MyD88, 36 STING may associate with the membrane proximal/cytoplasmic domain of MHC II, probably through CD79, in APC cells. 41 STING was also required for Fig. 1 Membrane bound CD4 in T cells controlled TLR4 inflammation. a Survival rates, (b) CD4 + T cell counts in the indicated organs, and (d) serum TNF and IL-6 were measured 12 h after i.p. LPS (n = 7-8). c Survival rates were measured after CLP (n = 7-16). e Absolute numbers of peripheral CD4 + T cells, (f) indicated cytokines of BALF were measured in COVID-19 patients (n = 15). Survival rates and TNF/IL-6 levels were measured as in (a-c) except that CD4 + T cells were (g) pre-depleted (GK1.5) for 2 days, or (h) supplemented to nude mice for 7 days, before LPS injection. i-k hACE2 mice pre-treated with GK1.5 antibody or isotype IgG were infected with SARS-CoV-2 for 7days. i Fold changes of TNF and IL-6 mRNA normalized to actin in the lung by qPCR and (j-k) representative sections and pathology score of the lobe of left lung (with respective magnifications of areas of interest) on day 7. SARS-CoV-2 virus (pfu 1 × 10 2 in box; pfu 1 × 10 5 in circle). l, m Measurement of TNF and IL-6 in supernatants of BMDM 16 h after incubation with LPS (100 ng/mL), in the absence or presence of (l) T cells of the indicated origins (macrophages: T cells = 1:1), or (m) naive CD4 + T cells with the indicated blocking mAb against CD4 (RM4-5; GK1.5). n TNF and IL-6 in supernatants 3 h after LPS treatment of THP-1 cells co-cultured with HeLa or TZM-B1 cells. Mean ± SD are shown; n = 3-11 mice used where indicated; Statistics (ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001): d Proportions of indicated cell types after in vitro differentiation of BM-derived monocytes co-cultured with CD4 + T cells, for the indicated time of LPS treatment. Cell numbers of (e) infiltrated monocyte derived macrophages or (g) resident macrophages and (f, h) their activation status (CD86 + ). i TNF mRNA induction by LPS in the indicated cell types sorted from the spleen. Mean ± SD are shown; n = 3-5 mice used where indicated; Statistics (ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001): Unpaired t test CD4/MHC II reverse signaling, since sCD4 no long improved the survival rates of LPS sepsis (Fig. 5j), nor inhibited TLR4 inflammation in eithr STING −/− mice (Fig. 5k) or STING −/− macrophages in vitro (Fig. 5l). Together, these results suggested that SHP2 and STING might bridge CD4/MHC II inhibitive crosstalk to TLR4/ MyD88 pro-inflammatory signaling.
sCD4 engagement dissociated the membrane raft domain of MHC II and TLR4 Because the cytoplasmic tail of MHC II is short and devoid of ITIM or ITAM motif, it was critical to test whether and how STING and/ or SHP2 directly tethered to MHC II. Duolink proximity ligation assays (PLA) were performed to measure these potential intermolecular interactions (Fig. 6a, red dots). At the resting state of cultured BMDM, SHP2 and STING, including activated SHP2 (pY580), were associated with MHC II. The interactions between MHC II and TLR4 in the cytoplasmic membrane, and MHC II with STING oligomerized in ER/Golgi 42 activated by LPS, were effectively disrupted by sCD4 (Fig. 6a). LPS dissociated the activated SHP-2 with MHC II, which could be effectively reverted by sCD4 (Fig. 6a). Merge of fluorescent antibody labeled MHC II (red) with Duolink dots of SHP2/STING (green) showed that SHP-2 and STING could form complex and were in close proximity to MHC II, and sCD4 reduced the assembly of MHCII-SHP2/STING complex (Fig. 6b). Furthermore, sCD4-increased pSHP-2/MHC II interaction was abolished in TLR4 −/− BMDM ( Supplementary Fig. 7a), suggesting that SHP-2, probably through complexing with STING, function to couple MHCII-TLR4 crosstalk for sCD4. These results would suggest that sCD4 dissociated LPS/TLR4 from MHC II/STING complex with presumably assistance of the activated SHP2.    6 sCD4 disrupted MHCII/TLR4 rafts and reduced LPS/TLR4 inflammatory membrane confinement. Duolink assays to quantify proteinprotein interactions of (a) the indicated pairs (red), or (b) between STING and SHP2 (green) that combined with immunofluorescent staining of MHC II (red) in BMDM. Tripartite colocalization indicated in yellow. The nuclei counter-stained with DAPI. Pearson's coefficients indicated the degree of colocalization. Bar = 5 μm. c TNF and IL-6 in supernatants 30 min after peritoneal macrophages treated with LPS or LPS plus sCD4, in the presence of indicated endocytosis inhibitors. The average of two independent repeats. Three replicate wells were used for each condition where the indicated inhibitor was added. d Duolink spots of MHC II-SHP2 interactions and (e) flow cytometric analyses of cell surface MHC II. Bar = 5 μm. Macrophages (5 x 10 6 ) were treated with LPS/sCD4 for 1 h, and (f) endosomes were isolated for immunoblot analysis of the indicated proteins (asterisk), or (g) organelle numbers per cell were quantified after immunofluorescence staining of EEA1 (early endosomes), LAMP1 (lysosomes), RAB4 (recycling endosome) and RCAS1 (Golgi). h Lysosomes size was quantified using LysoTracker after RAW264.7 cells were transfected with GFP-tagged MHC II Aβ or MHC II AβΔCT. Several view fields were randomly selected and images were acquired every 10 s for 20 min of LPS or LPS plus sCD4 treatment. Mean ± SD are shown; n = 3-4 mice used where indicated; Statistics (ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001;): Unpaired t test LPS confines TLR4 in membrane rafts so as to transmit proinflammatory signaling. 30,43 Inhibitors of lipid raft-(MCD) or clathrin-(Dynasore) mediated endocytosis impaired the ability of sCD4 to antagonize TLR4 inflammation (Fig. 6c) or dissociate SHP2 from MHC II (Fig. 6d). Therefore, the pro-inflammatiory LPS/TLR4 raft compartments might be disrupted by sCD4 engagement of MHCII membrane domains. On the other hand, sCD4 abrogated MHC II upregulation in BMDM surface by LPS, both by fluorescence staining (Fig. 6e and Supplementary Fig. 7b) and and flow cytometry detection ( Supplementary Fig. 7c), suggesting that sCD4 engagement might augment MHC II internalization [44][45][46] and/or lysosomal targeting. 46 LPS reduced overall the formation of early endosomes (Rab5), and concomitant endosomal distribution of MHC II, SHP2 and STING. sCD4 treatment, however, significantly restored their endosome trafficking (Fig. 6f). In addition to increased early endosome formation (EEA1), sCD4 also accelerated LAMP1-stained lysosome formation ( Fig. 6g and Supplementary  Fig. 7d) of MHC II complex. Live cell imaging indicated that sCD4driven endosome-lysosome fusion relied on the cytoplasmic domain of MHC II-Aβ (Fig. 6h), in agreement with the previous finding that MHC II cytoplasmic domain controls its endocytotic presentation. 47 Therefore, these results suggested that sCD4 disrupted pro-inflammation raft domains of MHC II/TLR4 complex, and by augmenting MHC II endocytosis, to destabilize proinflammatory membrane confinement of TLR4.

DISCUSSION
T lymphopenia occurs in the early phase of septic hyperinflammation, 16 is commonly associated with morbidity and mortality of septic infections. 17 Whether and how such a stoichiometric control of innate hyperinflammation is rooted in numerical reduction of naive CD4 + T cells, or CD4 molecule per se, remains elusive. This work establishes unambiguously that systemic hyperinflammtion upon infection was aggrevated by the loss of CD4 + T cells, that unleashed the restrains of MHC II/TLR4 inflammatory "signalosome" for cytokine storm or cytokine release syndrome in sepsis. This conclusion does not intend to exclude the contribution of CD8 + T lymphopenia in sepsis also observed in LPS and CLP models. Previous report showed that CD8 + T cells are able to engage MHC I to inhibit TLR inflammation. 40 CD4 molecule has been extensively studied, both structurally and functionally, as a co-receptor of TCR in facilitating MHC IIrestricted T-cell recognition. 48 CD4 contacts a conserved membrane-proximal region of MHC II, an action independent of TCR. 49 It has long been established that CD4/TCR complex uses MHC II as a receptor to feedforward activate antigen presentation signaling. 50 However, whether and how CD4/MHC II engagement negatively regulates APC inflammatory response to invading pathogens has never been addressed before. We revealed in this work that, CD4 molecule may function as an inhibitory ligand of MHC II, independent of TCR or CD40L, to constrain TLR inflammatory activation of resident macrophages, rather than monocyte-derived macrophages that had infiltrated to the lesion. CD4/MHC II engagement to downregulate TLR inflammation did not involve APC apoptosis, apparently different from the actions of antibodies or cognate TCR engagement of MHC II that accelerate cell death of activated DC, 51,52 macrophages 53,54 and B cells, 55,56 in caspase-independent, PKC/ERK-dependent manner. This and the previous work 8,57 also showed that TCR engagement is not essential for naive CD4 + T cells to inhibit PICC response in APC. Of course, CD4 + T cells may utilize co-inhibitory molecules, BTLA 58 or CD40L, 34 to down-regulate TNF response to LPS. CD40L signaling in effector/memory CD4 + T cells, independent of TCR, inhibits IL-1β secretion by APCs. 10 Other TNF superfamily members, RANKL, LIGHT, 41BBL, CD30L and OX40L, may also be involved in such an inhibitory T-APC immunological synapse. This work further show that CD4 molecule functions independent of CD40L, and not involving TNFR signaling, that essentially regulates the homeostatic APC inflammatory response.
Previous studies using antibody cross-linking, 59 super antigens 60,61 or lymphocyte-activating gene-3 (LAG-3), a MHC II ligand, 62 demonstrate that the heterodimeric MHC II protein complex can function as a receptor to activate T-dependent B cell response. In this context, an array of trans-membrane proteins, including CD79, CD19, CD20, CD21, CD40 and among others, physically associate with MHC II for B cell activation and differentiation. 50 Intriguing enough, we showed additionally that SHP2 activation and association with MHC II cytoplasmic tail, but not SHP1, was required for CD4 inhibition of TLR4/ MyD88 signaling and IRAK1/Erk activation. STING/SHP2 interaction has been shown in cytosolic DNA activated JAK1/ STAT1 signaling. 63 This is in line with previous observations that STING promotes MHC II-aggregation induced B lymphoma cell death and Erk activation independent of SHP1, 64 albeit SHP1 can associate with STING. 65 SHP2 also inhibits TLR3/TRIF signaling pathway in human monocytes. 66 It remains to test whether CD4 inhibition of TLR3 inflammation requires SHP2. Furthermore, sCD4/MHC II engagement did not alter LPS-induced phosphorylation of Btk, different from intracellular MHC II, which can interact and activate Btk via CD40 to promote endosomal TLR signaling. 67 Therefore, it is tempting to speculate that cell surface and intracellular MHC II may use different mediators to sort TLR signaling.
Importantly, we showed that increased serum sCD4 associated with LPS hyperinflammation. sCD4, presumably shed by a MMPlike sheddase, is also increased in patients of chronic inflammatory diseases, and correlates positively with the disease activities and poor prognosis in RA patients. 68 This would emphasize a compensatory effect of the increased sCD4 in control of inflammation, albeit sCD4 at sub-nanomolar concentrations in these patients is insufficient to do so. The binding affinity of CD4 ectodomain to pMHC II (~150-200 μM) is relatively weak as measured in vitro. 69 This far exceeds the concentrations of sCD4 needed for 50% inhibition of TLR4 hyperinflammation in vitro (~25 nM) or in vivo (~125 nM). We reason that auxiliary factors assembled within MHC II/TLR compartments in macrophages might help offset the low affinity of CD4 to MHC II. The exact makeup and function of MHC II/TLR rafts targeted by CD4 in this context remain to be determined. At least, TNFR is not associated with MHC II/TLR 'signalosome'. A nanomolar IC 50 would potentially make sCD4 (~10-15 mg/kg) especially desirable to prevent the onset of fatal sepsis. This work thus indicated that sCD4 might provide a new antiphlogistic paradigm to prevent severe/critical SIRS or sepsis. Applying sCD4 protein to target inflamed innate cells is apparently advantageous over the current therapeutics, 70 e.g., corticosteroid or antibodies, that target PICC or their cognate receptors ( Supplementary Fig. 9).

Mice
Mice homozygous null for MHC II genes (B6.129S-H2 dlAb1-Ea /J) were kindly provided by Dr. Xue-tao Cao (National Key Laboratory of Medical Immunology, Naval University of Medicine, Shanghai, China), SHP-2 fl/fl and LysM-cre knock-in mice by Drs. Hui Xiao and Gen-Sheng Feng, STING −/− mice by Dr. Xin-wen Chen (Wuhan Institute of Virology, Chinese Academy of Sciences). K18-hACE mice (C57BL/6 background) were purchased from Gem Pharmatech (Jiangsu, China). CD40-deficient mice were generated by CRISPR-Cas9 approach. Guide RNA sequences were listed in Supplementary Table 2. Littermates of 6-8 weeks (body weight and gender matched) were used, unless otherwise mentioned. Where used, nude mice, BABL/c and C57BL/6J mice were from Vital River Lab Animal Tech (Beijing). All mice were bred under specific pathogen-free conditions, founder mice and breeding Soluble CD4 effectively prevents excessive TLR activation of resident. . . Zhang et al. littermates were genotyped by PCR (primers in Supplementary  Table 3, and results in Supplementary Fig. 8). All animal experiments were performed in accordance with institutional guidelines and were approved by the Animal Care and Use Committees of the Institute of Biophysics or Institut Pasteur of Shanghai, Chinese Academy of Sciences (No. A2020043). Mouse models of sepsis The endotoxemia mouse model was established by i.p. injection of LPS (8-10 mg/kg body weight) in either C57BL/6J or nude mice. Where indicated, mice were i.p. injected with sCD4 (10 mg/kg) 12 h before LPS i.p. injection or transferred with CD4 + T cells 7 days before LPS injection. The cecal ligation and puncture (CLP) induced polymicrobial sepsis were performed as previously described. 71 Briefly, the mice were anesthetized, and a small midline abdominal incision was made. The cecum was then exteriorized, and the distal three-quarters of the cecum was immediately ligated without causing intestinal obstruction. The ligated cecum was punctured with an 18-gauge needle, and a small amount of feces was gently squeezed out of the perforation to ensure patency of the punctures. The cecum was then relocated into the abdominal cavity, and the incision was closed.

Reagents and antibodies
In the sham surgical controls, the cecum was exposed without ligation or puncture. At the indicated time points, mice were euthanized and the intestine, thymus, liver, spleen, and peripheral blood were collected for analysis. Thymus were fixed in 4% PFA solution and for histological stainings.
Histology Left lobes of lung or thymus were fixed in 4% paraformaldehyde and embedded in paraffin. Fixed tissues were sliced into 5 μm thick sections and stained with hematoxylin and eosin (H&E). To obtain a whole slide image, the slides with Mount coverslip onto the section on glass slide with neutral resins and subsequently the imaging data were obtained with Vectra3 with 20× objective (PerkinElmer, Vectra 3). Degrees of pathological alteration were scored following the standards described in Supplementary  Table 4.

Salmonella typhimurium infection
Salmonella typhimurium (strain SL1344) was grown overnight at 37 o C and then subcultured the next day in fresh LB to OD 600 = 0.6-0.8 at 37 o C. Water and food were withdrawn 4 h before gavage, mice (C57/B6j, male, 8 weeks old) were inoculated with 1 × 10 8 bacteria by oral gavage. Drinking water and food were supplied immediately. After 60 h, mice were euthanized and the blood, and spleen were harvested for bacterial loads, cytokines and immune cells analyses. All patients were hospitalized at Shenzhen Third People's Hospital 1 to 16 d after symptom onset. Among these patients, 11 had exposure history after traveling to Wuhan and other 4 had direct contact with COVID-19 patients from Wuhan. All patients presented with fever, fatigue and dry cough, and 5 had developed severe pneumonia when admitted to the hospital (patient number C3, C4, C8, C11, C14). Eleven patients had underlying disease such as hypertension, cardiopathy, autoimmune disorder or chronic hepatitis B. All patients progressed from mild to severe pneumonia, or severe to mild pneumonia, and all recovered and discharged after symptomatic treatment. Patient C11 succumbed to COVID-19 in hospital. All patients received interferon and ribavirin and lopinavir and/or methylprednisolone treatments.
SARS-CoV-2 infection of PBMC PBMC (5 × 10 5 cells/ml) were seeded in 96-well plate with RPMI-1640 for 12 h before incubation with 50 nM sCD4 for additional 2 h. Cells were then infected with SARS-CoV2 (separated from patient YCJ. MOI = 0.1 or 0.5, respectively). Supernatants were collected after 24 h of infection, and the virus was inactivated using β-lactone (Solarbio) at 4 o C for 36 h. TNF and IL-6 were measured by by using Elisa kit (BioLegend). Experiment perfomed in a Bio-safety Level 3 facility. Flow cytometry DN T cells, monocytes and macrophages in the spleen, or DN T and SP CD4 + T cells in the thymus were isolated (1 × 10 7 splenocytes/thymocytes labeled with 5 μg indicated antibodies in 1 mL staining buffer (PBS, 1% FBS)) by FACSAria II sorter (BD Biosciences). Multicolor flow cytometric analyses were routinely performed on LSR Fortessa (BD Bioscience) and analyzed by FlowJO software (Tree Star, OR). Dead cells were excluded with 7-AAD (eBioscience) or DAPI (BD Biosciences). Detailed antibodies information in Supplementary Table 5. Analysis of apoptosis was performed with Annexin V-FITC Apoptosis Detection Kit according to the manufacturer's instruction (BD Biosciences).

ELISA analysis of serum sCD4
To measure mourse CD4 in sera, 96-well high binding plates (NEST) were coated with anti-CD4 (GK1.5, BioLegend), followed by an incubation with 10 μL two-fold serially diluted samples to be tested. After washing, the wells were incubated with biotinylated anti-CD4 (RM4-4, BioLegend) and subsequently with HRP-coupled streptavidin. The levels of sCD4 were photometrically determined according to the manufacturer's manual (Sino Biological). The results are presented as mean ± SEM of at least three samples.
Immunoblot and immunoprecipitation Where indicated, membrane proteins were extracted using Minute Plasma Membrane Protein Isolation and Cell Fractionation Kit (SM-005; Invent). Whole cell lysates were routinely prepared as previously described, 72 and protease inhibitors cocktail (Roche) added before immunoprecipitation. Total protein concentrations measured by BCA assay (Pierce).
Duolink proximity ligation assay (PLA) Peritoneal macrophages (PM) or BMDM cells (6 × 10 5 ) were seeded on a cover slip in a 6-well plate, and serum-starved in OPTI-MEM overnight, LPS or/and sCD4 were then added at the indicated concentrations for 30 min. Cells were then fixed (4% PFA, 4 o C, 15 min), quenched (0.1 M glycine in PBS, 4 o C, 10 min), washed twice with cold PBS. Permeabilization step was applied for detection of cytosolic proteins (saponin solution, #P0095, Beyotime), 30 min). Cells were blocked using Doulink in situ RED starter kit (Sigma, DUO92101-1KT) for 1 h at room temperature. Primary antibodies (1 μg/mL) targeting MHCII, TLR4, SHP-2, pSHP-2) were used and inter-protein interaction was visualized with probes (DUO92101-1KT, Sigma) that binds to the primary antibodies according to the manufacturer's protocol. Images were captured with DeltaVison microscope (60x, oil; GE), and red fluorescent dots representative of 1 inter-molecular interaction were counted (4 view fields and at least 160 cells) and analyzed with Imaris Software (Oxford Instruments). IgG isotype used as control.
Immunofluorescence staining Peritoneal macrophages (3 × 10 5 ) were seeded on a cover slip in a 6-well plate, and serum-starved overnight in OPTI-MEM before LPS or/and sCD4 were added. Cells were then fixed, quenched and washed as PLA assays, except that 0.25% Triton X-100 was used for permeabilization, and 1% BSA in PBST for blocking. Primary antibodies against MHCII, TLR4, EEAL, LAPM1, RAB4, RCAS1, and secondary antibodies (Goat anti-Rat Alexa Fluor 647; Donkey anti-Mouse IgG Alexa Fluor 488; Goat anti-Rabbit Alexa Fluor 555) were applied for fluorescent imaging analyses (DeltaVision OMX, 60 x, oil; GE). Green fluorescent dots (10 view fields and at least 10 cells) were counted and analyzed with Imaris Software (Oxford Instruments).
Live cell imaging of lysosomes RAW264.7 cells (1 × 10 5 ) were seeded in a glass bottom dish (35 mm) for 24 h, then transfected with GFP tagged MHC IIAβ or MHC IIAβΔCT. 24 h later, cells were spiked with lysosome tracker for 30 min. Washed and replaced with pre-warmed fresh medium, LPS (500 ng/mL) or LPS plus sCD4 (125 nM) added to cells. Live cell imaging (every 10 s for 20 min) of 4 random view fields were acquired using Olympus SpinSR10 Ixplore microscope (60x, oil). The size of the lysosome was analyzed by the Surface combined with the automatically tracking objects of interest using the provided algorithms based on Imaris Software (Oxford Instruments).
Statistical analysis Statistical analysis was performed by using Log-rank (Mantel-Cox) test, Unpaired t test and one-way ANOVA with Dunnett's analysis (Graphpad Prism 8). Data are presented as the mean ± SD. Pvalue < 0.05 was considered statistically significant.

DATA AVAILABILITY
Data that support the findings of this study are available upon reasonable request to the lead contact (H.T.).